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Activation of mitogen-activated protein kinases (MAPKs) is one of the earliest responses after plants sense an invading pathogen. Here, we show that MPK3 and MPK6, two Arabidopsis thaliana pathogen-responsive MAPKs, and their upstream MAPK kinases, MKK4 and MKK5, are essential to both stomatal and apoplastic immunity. Loss of function of MPK3 and MPK6, or their upstream MKK4 and MKK5, abolishes pathogen/microbe-associated molecular pattern- and pathogen-induced stomatal closure. Gain-of-function activation of MPK3/MPK6 induces stomatal closure independently of abscisic acid (ABA) biosynthesis and signaling. In contrast, exogenously applied organic acids such as malate or citrate are able to reverse the stomatal closure induced by MPK3/MPK6 activation. Gene expression analysis and in situ enzyme activity staining revealed that malate metabolism increases in guard cells after activation of MPK3/MPK6 or inoculation of pathogen. In addition, pathogen-induced malate metabolism requires functional MKK4/MKK5 and MPK3/MPK6. We propose that the pathogen-responsive MPK3/MPK6 cascade and ABA are two essential signaling pathways that control, respectively, the organic acid metabolism and ion channels, two main branches of osmotic regulation in guard cells that function interdependently to control stomatal opening/closure.

Background Ferroptosis, a new form of programmed cell death, has great potential for cancer treatment. However, the roles of ferroptosis-related (FR) genes in breast cancer (BC) remain elusive. Materials and methods Using TCGA database, a novel FR risk signature was constructed through the Lasso regression analysis. Meanwhile, its prognostic value was assessed by a series of survival analyses. Besides, a nomogram was constructed to predict the overall survival rate (OSR) of individual at 1,3,5 year. Four validation cohorts ( n  = 2248), including METABRIC, GSE58812, GSE20685 and ICGC-KR datasets, were employed to test the prognostic value of FR risk signature. The effects of FR risk signature on BC immune microenvironment were explored by CIBERSORT algorithm and ssGSEA method. The histological expressions of FR risk genes were presented by HPA database. The biofunctions of SQLE were determined by qPCR, MTT, wound-healing and Transwell assays. Results We constructed a novel FR risk signature consisting of eight genes. High FR risk led a poor prognosis and was identified as an independent prognostic factor. Besides, A higher proportion of patients with luminal A type was observed in low-risk group (53%), while a higher proportion of patients with basal type in high-risk group (24%). FR risk score could discriminate the prognostic difference of most clinical subgroups, except for M1 stage, HER2 and basal types. Moreover, its prognostic value was successfully validated in other four cohorts. Through immune analyses, we found that the reduced infiltration levels of CD8+ and NK cells, whereas the enhanced activity of antigen presentation process appeared in high FR risk. Then, FR risk score was found to weakly correlate with the expressions of six immune checkpoints. Through the experiments in vitro, we confirmed that overexpression of SQLE could promote, whereas blocking SQLE could inhibit the proliferative, migrative and invasive abilities of BC cells. Conclusions FR risk signature was conducive to BC prognostic assessment. High FR risk level was closely associated with BC immunosuppression, but may not predict ICIs efficacy. Moreover, SQLE was identified as a crucial cancer-promoting gene in BC. Our findings provide new insights into prognostic assessment and molecular mechanism of BC.

Cytokines that stimulate T cell proliferation, such as interleukin (IL)-15, have been explored as a means of boosting the antitumor activity of chimeric antigen receptor (CAR) T cells. However, constitutive cytokine signaling in T cells and activation of bystander cells may cause toxicity. IL-23 is a two-subunit cytokine known to promote proliferation of memory T cells and T helper type 17 cells. We found that, upon T cell antigen receptor (TCR) stimulation, T cells upregulated the IL-23 receptor and the IL-23α p19 subunit, but not the p40 subunit. We engineered expression of the p40 subunit in T cells (p40-Td cells) and obtained selective proliferative activity in activated T cells via autocrine IL-23 signaling. In comparison to CAR T cells, p40-Td CAR T cells showed improved antitumor capacity in vitro, with increased granzyme B and decreased PD-1 expression. In two xenograft and two syngeneic solid tumor mouse models, p40-Td CAR T cells showed superior efficacy in comparison to CAR T cells and attenuated side effects in comparison to CAR T cells expressing IL-18 or IL-15. The efficacy of chimeric antigen receptor (CAR) T cells in solid tumor models is enhanced by interleukin-23 engineering.

The triple negative breast cancer (TNBC) is the most malignant subtype of breast cancer with high aggressiveness. Although paclitaxel-based chemotherapy scenario present the mainstay in TNBC treatment, paclitaxel resistance is still a striking obstacle for cancer cure. So it is imperative to probe new therapeutic targets through illustrating the mechanisms underlying paclitaxel chemoresistance. The Single cell RNA sequencing (scRNA-seq) data of TNBC cells treated with paclitaxel at different points were downloaded from the Gene Expression Omnibus (GEO) database. The Seurat R package was used to filter and integrate the scRNA-seq expression matrix. Cells were further clustered by the FindClusters function, and the gene marker of each subset was defined by FindAllMarkers function. Then, the hallmark score of each cell was calculated by AUCell R package, the biological function of the highly expressed interest genes was analyzed by the DAVID database. Subsequently, we performed pseudotime analysis to explore the change patterns of drug resistance genes and SCENIC analysis to identify the key transcription factors (TFs). Finally, the inhibitors of which were also analyzed by the CTD database. We finally obtained 6 cell subsets from 2798 cells, which were marked as AKR1C3+, WNT7A+, FAM72B+, RERG+, IDO1+ and HEY1+HCC1143 cell subsets, among which the AKR1C3+, IDO1+ and HEY1+ cell subsets proportions increased with increasing treatment time, and then were regarded as paclitaxel resistance subsets. Hallmark score and pseudotime analysis showed that these paclitaxel resistance subsets were associated with the inflammatory response, virus and interferon response activation. In addition, the gene regulatory networks (GRNs) indicated that 3 key TFs (STAT1, CEBPB and IRF7) played vital role in promoting resistance development, and five common inhibitors targeted these TFs as potential combination therapies of paclitaxel were identified. In this study, we identified 3 paclitaxel resistance relevant IFs and their inhibitors, which offers essential molecular basis for paclitaxel resistance and beneficial guidance for the combination of paclitaxel in clinical TNBC therapy.

Extensive research revealed tremendous details about how plants sense pathogen effectors during effector-triggered immunity (ETI). However, less is known about downstream signaling events. In this report, we demonstrate that prolonged activation of MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MPKs), is essential to ETI mediated by both coiled coil-nucleotide binding site-leucine rich repeats (CNLs) and toll/interleukin-1 receptor nucleotide binding site-leucine rich repeats (TNLs) types of R proteins. MPK3/MPK6 activation rapidly alters the expression of photosynthesis-related genes and inhibits photosynthesis, which promotes the accumulation of superoxide ([Formula: see text]) and hydrogen peroxide (H2O2), two major reactive oxygen species (ROS), in chloroplasts under light. In the chemical-genetically rescued mpk3 mpk6 double mutants, ETI-induced photosynthetic inhibition and chloroplastic ROS accumulation are compromised, which correlates with delayed hypersensitive response (HR) cell death and compromised resistance. Furthermore, protection of chloroplasts by expressing a plastid-targeted cyanobacterial flavodoxin (pFLD) delays photosynthetic inhibition and compromises ETI. Collectively, this study highlights a critical role of MPK3/MPK6 in manipulating plant photosynthetic activities to promote ROS accumulation in chloroplasts and HR cell death, which contributes to the robustness of ETI. Furthermore, the dual functionality of MPK3/MPK6 cascade in promoting defense and inhibiting photosynthesis potentially allow it to orchestrate the trade-off between plant growth and defense in plant immunity.

Plant sensing of invading pathogens triggers massive metabolic reprogramming, including the induction of secondary antimicrobial compounds known as phytoalexins. We recently reported that MPK3 and MPK6, two pathogen-responsive mitogen-activated protein kinases, play essential roles in the induction of camalexin, the major phytoalexin in Arabidopsis thaliana. In search of the transcription factors downstream of MPK3/MPK6, we found that WRKY33 is required for MPK3/MPK6-induced camalexin biosynthesis. In wrky33 mutants, both gain-of-function MPK3/MPK6-and pathogen-induced camalexin production are compromised, which is associated with the loss of camalexin biosynthetic gene activation. WRKY33 is a pathogen-inducible transcription factor, whose expression is regulated by the MPK3/MPK6 cascade. Chromatin immunoprecipitation assays reveal that WRKY33 binds to its own promoter in vivo, suggesting a potential positive feedback regulatory loop. Furthermore, WRKY33 is a substrate of MPK3/MPK6. Mutation of MPK3/MPK6 phosphorylation sites in WRKY33 compromises its ability to complement the camalexin induction in the wrky33 mutant. Using a phospho-protein mobility shift assay, we demonstrate that WRKY33 is phosphorylated by MPK3/MPK6 in vivo in response to Botrytis cinerea infection. Based on these data, we conclude that WRKY33 functions downstream of MPK3/MPK6 in reprogramming the expression of camalexin biosynthetic genes, which drives the metabolic flow to camalexin production in Arabidopsis challenged by pathogens.

Baicalein, a widely used Chinese herbal medicine, has shown anticancer effects on many types of human cancer cell lines. However, little is known about the underlying mechanism in human breast cancer cells. In this study, we examined the apoptotic and autophagic pathways activated following baicalein treatment in human breast cancer cells in vitro and in vivo. In in vitro study, we used MTT and clone formation assay to confirm the inhibitory role of baicalein on proliferation of MCF-7 and MDA-MB-231 breast cancer cells. Apoptosis was detected employing Hoechst 33258 staining, JC-1 staining, and flow cytometry. Autophagy was monitored by acridine orange staining and transmission electron microscopy observation. Quantitative real-time PCR and Western blot analysis were employed to study the effects of baicalein on PI3K/AKT signaling components of MCF-7 and MDA-MB-231 breast cancer cells. In in vivo study, the effect of baicalein was tested with a breast cancer cells transplantation tumor model. Our study showed that baicalein has the potential to suppress cell proliferation, induce apoptosis and autophagy of breast cancer cells in vitro and in vivo. Furthermore, baicalein significantly downregulated the expression of p-AKT, p-mTOR, NF-κB, and p-IκB while enhancing the expression of IκB in MCF-7 and MDA-MB-231 cells. It also decreased the p-AKT/AKT and p-mTOR/mTOR ratios. Our study demonstrated that baicalein induces apoptosis and autophagy of breast cancer cells via inhibiting the PI3K/AKT signaling pathway in vivo and vitro. Our study revealed that baicalein may be a potential therapeutic agent for breast cancer.

Plant defense often depends on the synthesis and targeted delivery of antimicrobial metabolites at pathogen contact sites. The pleiotropic drug resistance (PDR) transporter PENETRATION3 (PEN3)/PDR8 in Arabidopsis (Arabidopsis thaliana) has been implicated in resistance to a variety of fungal pathogens. However, the antimicrobial metabolite(s) transported by PEN3 for extracellular defense remains unidentified. Here, we report that PEN3 functions redundantly with another PDR transporter (PDR12) to mediate the secretion of camalexin, the major phytoalexin in Arabidopsis. Consistent with this, the pen3 pdr12 double mutants exhibit dramatically enhanced susceptibility to the necrotrophic fungus Botrytis cinerea as well as severe hypersensitivity to exogenous camalexin. PEN3 and PDR12 are transcriptionally activated upon B. cinerea infection, and their expression is regulated by the mitogen-activated protein kinase 3 (MPK3) and MPK6, and their downstream WRKY33 transcription factor. Further genetic analysis indicated that PEN3 and PDR12 contribute to B. cinerea resistance through exporting not only camalexin but also other unidentified metabolite(s) derived from Trp metabolism, suggesting that PEN3 and PDR12 have multiple functions in Arabidopsis immunity via transport of distinct Trp metabolic products.

Plants under pathogen attack produce high levels of ethylene, which plays important roles in plant immunity. Previously, we reported the involvement of ACS2 and ACS6, two Type I ACS isoforms, in Botrytis cinerea-induced ethylene biosynthesis and their regulation at the protein stability level by MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MAPKs). The residual ethylene induction in the acs2/acs6 double mutant suggests the involvement of additional ACS isoforms. It is also known that a subset of ACS genes, including ACS6, is transcriptionally induced in plants under stress or pathogen attack. However, the importance of ACS gene activation and the regulatory mechanism(s) are not clear. In this report, we demonstrate using genetic analysis that ACS7 and ACS11, two Type III ACS isoforms, and ACS8, a Type II ACS isoform, also contribute to the B. cinerea-induced ethylene production. In addition to post-translational regulation, transcriptional activation of the ACS genes also plays a critical role in sustaining high levels of ethylene induction. Interestingly, MPK3 and MPK6 not only control the stability of ACS2 and ACS6 proteins via direct protein phosphorylation but also regulate the expression of ACS2 and ACS6 genes. WRKY33, another MPK3/MPK6 substrate, is involved in the MPK3/MPK6-induced ACS2/ACS6 gene expression based on genetic analyses. Furthermore, chromatin-immunoprecipitation assay reveals the direct binding of WRKY33 to the W-boxes in the promoters of ACS2 and ACS6 genes in vivo, suggesting that WRKY33 is directly involved in the activation of ACS2 and ACS6 expression downstream of MPK3/MPK6 cascade in response to pathogen invasion. Regulation of ACS activity by MPK3/MPK6 at both transcriptional and protein stability levels plays a key role in determining the kinetics and magnitude of ethylene induction.

Arabidopsis thaliana MPK3 and MPK6, two mitogen-activated protein kinases (MAPKs or MPKs), play critical roles in plant disease resistance by regulating multiple defense responses. Previously, we characterized the regulation of phytoalexin biosynthesis by Arabidopsis MPK3/MPK6 cascade and its downstream WRKY33 transcription factor. Here, we report another substrate of MPK3/MPK6, ETHYLENE RESPONSE FACTOR6 (ERF6), in regulating Arabidopsis defense gene expression and resistance to the necrotrophic fungal pathogen Botrytis cinerea. Phosphorylation of ERF6 by MPK3/MPK6 in either the gain-of-function transgenic plants or in response to B. cinerea infection increases ERF6 protein stability in vivo. Phosphomimicking ERF6 is able to constitutively activate defense-related genes, especially those related to fungal resistance, including PDF1.1 and PDF1.2, and confers enhanced resistance to B. cinerea. By contrast, expression of ERF6-EAR, in which ERF6 was fused to the ERF-associated amphiphilic repression (EAR) motif, strongly suppresses B. cinerea—induced defense gene expression, leading to hypersusceptibility of the ERF6-EAR transgenic plants to B. cinerea. Different from ERF1, the regulation and function of ERF6 in defensin gene activation is independent of ethylene. Based on these data, we conclude that ERF6, another substrate of MPK3 and MPK6, plays important roles downstream of the MPK3/MPK6 cascade in regulating plant defense against fungal pathogens.